Breast-feeding is recommended to prevent the development of allergic diseases, but mechanisms are unclear. Human milk is a source of human milk oligosaccharides (HMOs) and fatty acids (FAs), which are likely impacted by maternal diet. HMOs are complex glycans prevalent in human milk; non-digestible by humans, but one of the main substrates for the infant gut microbiota. We have recently shown that mothers' milk has significant variations in qualitative and quantitative HMO composition, depending on genetics and maternal probiotic supplementation. Human milk is a rich source of FAs such as polyunsaturated (PUFAs) and short-chain fatty acids (SCFAs); these are strongly influenced by maternal diet and could directly induce mucosal immunity. The impact of maternal diet and supplements on immunomodulatory composition of human milk is poorly characterized and varies between mothers. Our initial studies have shown that certain HMOs are associated with protection against cow's milk allergy in the infant, but the mechanisms are not known. Our initial study in the Old Order Mennonites (OOM), who have a lifestyle dating back to 150 years ago and rich exposure to unpasteurized milk, indicated a low prevalence of asthma and allergic diseases, compared to the Rochester population. OOM mothers also have rich sources of diet-based probiotics such as unpasteurized dairy products (kefir, yogurt, cheese, butter); these can significantly affect their breast milk FA and HMO composition, whereas OTC supplement use may not be common. Our central hypothesis is that factors related to OOM diet decrease the risk of allergies through impact on human milk composition. Our preliminary data suggests that the infant gut microbiome is enriched in Bifidobacteria in the OOM. Bifidobacteria in turn are largely impacted by human milk composition. The overall objective of this supplemental application is to expand our ongoing longitudinal infant cohort study, entitled ?Mucosal and systemic immunity and food allergy? (U01 AI131344), by gathering and analyzing samples to define the role of maternal diet, including dietary and OTC probiotics on human milk composition, and to assess their impact on infant immunity. The parent study is characterizing the infant gut microbiome, immune markers and atopic diseases in the first year of life in OOM and Rochester infants. We have a unique opportunity to investigate maternal dietary impact on human milk composition. This additional data along with modeling as part of the parent grant will provide holistic understanding of interactions between OOM lifestyle and infant gut microbiome composition, immune markers and allergic disease outcomes in infants. 1